TSG-Like Gene

ABSTRACT

A gene encoding a novel protein that is homologous to  Drosophila  TSG was isolated from a cDNA library derived from the AGM region of mouse embryos by using an originally developed cloning method specific to a gene encoding a membrane secretory protein. This gene is useful in developing drugs that regulate hematopoietic stem cell generation, immune and hematopoietic functions, etc.

This application is a continuation of U.S. Ser. No. 11/079,947, filedMar. 15, 2005, which is a divisional application of U.S. Ser. No.10/092,925, filed Mar. 6, 2002, now U.S. Pat. No. 6,890,735, which is acontinuation-in-part of International Patent Application No.PCT/JP00/06050, filed Sep. 6, 2000, which claims priority to JapanesePatent Application No. 11-252190, filed Sep. 6, 1999. The disclosures ofall of these applications are herein incorporated by reference.

TECHNICAL FIELD

The present invention relates to a novel TSG-like protein and its genederived from the AGM region of mouse embryos.

BACKGROUND

In the early fetal period of mice, hematopoiesis is carried out in theyolk sac and fetal liver. Hematopoiesis in the yolk sac is referred toas fetal hematopoiesis during which primarily nucleated fetalerythrocytes are produced. On the other hand, hematopoiesis in the fetalliver is referred to as adult hematopoiesis during which all lines ofblood cells are produced with the exception of nucleated fetalerythrocytes.

Although the activity that causes the production of all lines of bloodcells, that is the long-term repopulating hematopoietic stem cell(LTR-HSC) activity of hematopoietic stem cells, is not detected in fetalhematopoiesis, it is detected in adult hematopoiesis. It is now thoughtthat the cells having this LTR-HSC activity are actually produced notinitially in the liver, but in the aorta-gonad-mesonephros (AGM) regionat day 10-11 of embryogenesis. During this period, these cells arethought to also proliferate in this AGM region, after which they migrateto the fetal liver (Medvinsky et al., Cell 86:897-906). Thus, a genethat is important for the generation of hematopoietic stem cells may beexpressed in this AGM region.

SUMMARY

The present invention provides a novel TSG-like protein and its genederived from the AGM region of mouse embryos. In addition, the presentinvention also provides a vector into which the gene is inserted, a hostcell carrying the vector, and an antibody that binds to the protein.Moreover, the present invention provides a method for screeningcompounds, such as receptors, that bind to the protein by using theprotein.

The present inventors screened cDNA that encode secretory/membraneproteins to search for a gene having a novel signal sequence from theAGM region of mouse embryos, using poly(A) RNA derived from this AGMregion as the starting material, and an originally-developed signalsequence trap (SST) method (Japanese Patent Application No. Hei9-324912). As a result, the present inventors succeeded in isolating agene that encodes a novel protein homologous to Drosophila TSG gene. TSGgene is one of the dorsal determining factors of an embryo, and is knownto determine differentiation of dorsal midline cells due to interactionwith DPP (the counterpart of BMP2/4) (Mason et al., Genes andDevelopment 8:1489-1501). Since TSG protein has been reported to bind toBMP (Oelgeschlager et al., Nature 405:757-763, 2000), the isolatedTSG-like gene, which is structurally similar to the TSG gene, ispredicted to interact with BMP2/4. In addition, the fact that thisTSG-like gene was isolated from the AGM region of mouse embryos suggestsits involvement in the generation of hematopoietic stem cells. Thus, theTSG-like protein of the present invention is useful as a tool forpurifying and screening factors involved in the generation ofhematopoietic stem cells, and the screening of drug candidate compoundsfor immune and hematopoietic system-related diseases.

The present invention relates to a novel TSG-like protein, its gene, aswell as the production and uses of the protein and gene. Morespecifically, the present invention relates to:

(1) a DNA according to any one of (a) to (d):

(a) a DNA encoding a protein comprising the amino acid sequence of SEQID NO:2,

(b) a DNA comprising the coding region of the nucleotide sequence of SEQID NO:1,

(c) a DNA comprising an amino acid sequence in which one or more aminoacids of the amino acid sequence of SEQ ID NO:2 has been substituted,deleted, inserted and/or added, wherein said DNA encodes a protein thatis functionally equivalent to the protein comprising the amino acidsequence of SEQ ID NO:2, and,

(d) a DNA hybridizing to a DNA that comprises the nucleotide sequence ofSEQ ID NO:1 under stringent conditions, and, encodes a proteinfunctionally equivalent to the protein comprising the amino acidsequence of SEQ ID NO:2;

(2) a DNA encoding a partial peptide of the protein comprising the aminoacid sequence of SEQ ID NO:2;

(3) a vector into which the DNA according to (1) or (2) has beeninserted;

(4) a transformant carrying the DNA according to (1) or (2) or thevector according to (3);

(5) a protein or peptide encoded by the DNA according to (1) or (2);

(6) a method for producing the protein or peptide according to (5),comprising the steps of culturing the transformed cell according to (4),and recovering the expressed protein from said cell or the culturesupernatant;

(7) an antibody against the protein according to (5);

(8) a oligonucleotide that

hybridizes to the DNA comprising of the nucleotide sequence of SEQ IDNO:1, or the complementary strand thereof, and,

comprises at least 15 nucleotides;

(9) a method of screening for a compound having the activity of bindingto the protein according to (5), comprising the steps of:

(a) contacting a test sample with the protein or partial peptideaccording to (5), and,

(b) selecting a compound having an activity of binding to the protein orpartial peptide according to (5); and,

(10) a compound isolated using a method as set forth in (9), having anactivity of binding to the protein according to (5).

The present invention relates to a novel protein that is homologous tothe Drosophila TSG gene. A mouse-derived cDNA nucleotide sequenceisolated by the present inventors is shown in SEQ ID NO:1, while theamino acid sequence of the protein encoded by the cDNA is shown in SEQID NO:2. This protein has a signal sequence at its N terminus, and ishomologous to TSG protein, a dorsal determining factor of the Drosophilaembryo. As a result of Northern blot analysis of mRNA derived from mousetissues, a signal of about 4.0 kb was observed in the heart, lung,liver, and kidney. In addition, this signal was also confirmed to beexpressed in 9, 10, 11, 12, and 13-day viviparity. The isolation fromthe AGM region of the embryo, expression in early embryos, homology toTSG protein and presumed interaction with BMP2/4, the fact that BMP2/4is required for the differentiation of blood cell lines, the fact thatTSG protein binds to BMP to promote signaling activity of BMP(Oelgeschlager et al., Nature 405:757-763, 2000), all suggest that thisprotein may be involved in the differentiation of hematopoietic cells aswell as bone formation, and so forth. Thus, this protein can be utilizedas a tool for purifying and cloning proteins related to hematopoieticstem cell formation, bone formation, and so forth, and for screeningdrug candidate compounds as therapeutic agents for immune andhematopoietic system-related diseases, bone formation-related diseases,and such.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. For example, the substantially purepolypeptide is at least 75%, 80, 85, 95, or 99% pure by dry weight.Purity can be measured by any appropriate standard method known in theart, for example, by column chromatography, polyacrylamide gelelectrophoresis, or HPLC analysis.

Accordingly, the invention includes a polypeptide having a sequenceshown as SEQ ID NO:2. The invention also includes a polypeptide, orfragment thereof, that differs from the corresponding sequence shown asSEQ ID NO:2. The differences are, preferably, differences or changes ata non-essential residue or a conservative substitution. In oneembodiment, the polypeptide includes an amino acid sequence at leastabout 60% identical to a sequence shown as SEQ ID NO:2, or a fragmentthereof. Preferably, the polypeptide is at least 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, 99% or more identical to SEQ ID NO:2 and has atleast one TSG-like function or activity described herein. Preferredpolypeptide fragments of the invention are at least 10%, preferably atleast 20%, 30%, 40%, 50%, 60%, 70%, or more, of the length of thesequence shown as SEQ ID NO:2 and have at least one TSG-like function oractivity described herein. Or alternatively, the fragment can be merelyan immunogenic fragment.

In addition, the present invention also includes proteins that arefunctionally equivalent to the protein described in SEQ ID NO:2. Suchproteins include, for example, homologous proteins of other organismscorresponding to the protein described in SEQ ID NO:2, as well asmutants of the protein. In the present invention, the term “functionallyequivalent” means that the target protein has an activity for rescuingaberrations in the differentiation of dorsal midline cells when injectedinto a TSG mutant of Drosophila, or an activity that regulates embryodevelopment (for example, dorsoventral induction capability) wheninjected into Xenopus eggs. In addition, the protein of the presentinvention is also suggested to have the function of promoting thesignaling activity of BMP (bone morphogenetic protein; DPP) by bindingwith BMP (Oelgeschlager et al., Nature 405:757-763, 2000).

One method for isolating such proteins well known to those skilled inthe art is to introduce mutations into the proteins. For example, oneskilled in the art can prepare proteins functionally equivalent to theprotein of SEQ ID NO:2 by introducing appropriate mutations into theamino acid of SEQ ID NO:2, by using site-specific mutagenesis(Hashimoto-Gotoh et al., Gene 152:271-275, 1995; Zoller et al., MethodsEnzymol. 100:468-500, 1983; Kramer et al., Nucleic Acids Res.12:9441-9456, 1984; Kramer et al., Methods Enzymol. 154:350-367, 1987;Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492, 1985; Kunkel MethodsEnzymol. 85:2763-2766, 1988). Mutation of amino acids may occur innature, too. The protein of the present invention also includes aprotein comprising the amino acid sequence of SEQ ID NO:2 in which oneor more amino acids are mutated, wherein the resulting “mutant” proteinis functionally equivalent to the protein of SEQ ID NO:2. In such amutant protein, the number of the amino acids mutated are considered tobe usually 30 residues or less, preferably 15 residues or less, morepreferably 5 residues or less, and still preferably, 3 residues or less.

The mutated amino acid residue is preferably mutated into an amino acidthat allows the properties of the amino acid side-chain to be conserved.Examples of properties of amino acid side chains include: hydrophobicamino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D,N, C, E, Q, G, H, K, S, T), and amino acids comprising the followingside chains: an aliphatic side-chain (Q A, V, L, I, P); a hydroxyl groupcontaining side-chain (S, T, Y); a sulfur atom-containing side-chain (C,M); a carboxylic acid- and amide-containing side-chain (D, N, E, Q); anucleotide-containing side-chain (R, K, H); and an aromatic-containingside-chain (H, F, Y, W) (the letters within the parentheses indicate theone-letter codes of amino acids).

It is well known that a protein having a deletion, addition, and/orsubstitution of one or more amino acid residues in the sequence of theprotein can retain the original biological activity (Mark et al., Proc.Natl. Acad. Sci. USA 81:5662-5666, 1984; Zoller et al., Nucleic AcidsRes. 10:6487-6500, 1982; Wang et al., Science 224:1431-1433;Dalbadie-McFarland et al., Proc. Natl. Acad. Sci. USA 79:6409-6413,1982).

A protein having the amino acid sequence of SEQ ID NO:2, to which one ormore amino acid residues have been added, is exemplified by a fusionprotein containing the protein of SEQ ID NO:2. Fusion proteins, in whichthe protein listed in SEQ ID NO:2, or its partial peptide is fused toother peptides or proteins, are included in the present invention.Fusion proteins can be made using well-known techniques by linking theDNA encoding the protein of the invention in frame with the DNA encodinganother peptide or protein, followed by inserting the DNA into anexpression vector, and expressing it in a host. There is no restrictionas to the peptides or proteins to be fused to the protein of the presentinvention.

Other known peptides that can be used for fusion with the protein of thepresent invention include, for example, FLAG (Hopp et al., BioTechnology6:1204-1210, 1988), 6×His comprised of six His (histidine) residues,10×His, influenza agglutinin (HA), human c-myc fragment, VSV-GPfragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigenfragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment, etc.In addition, examples of other proteins used for fusion with the proteinof the present invention include, for example, GST(glutathione-S-transferase), HA (influenza agglutinin), immunoglobulinconstant region, β-galactosidase, MBP (maltose binding protein), etc.

Fusion proteins can be prepared by fusing DNA encoding thesecommercially available peptides or proteins with DNA encoding theprotein of the present invention and expressing the prepared fused DNA.

An alternative method for isolating functionally equivalent proteinsknown to those skilled in the art is, for example, a method utilizingthe hybridization technique (Sambrook et al., Molecular Cloning 2nd ed.9.47-9.58, Cold Spring Harbor Lab. Press, 1989). Generally, one skilledin the art can isolate DNAs highly homologous to the whole or part ofthe DNA sequence encoding the protein of SEQ ID NO:2 or 4 (SEQ ID NO:1or 3, respectively), and then isolate a DNA that codes for a proteinfunctionally equivalent to the protein of SEQ ID NO:2 or 4 from the DNAisolated. The proteins of the present invention thus include proteinsencoded by DNA that hybridize with the whole or part of the DNA sequenceencoding the protein of SEQ ID NO:2 or 4, wherein the proteins arefunctionally equivalent to the protein of SEQ ID NO:2 or 4. Theseproteins include homologues from mammals except mice (for example, aprotein encoded by a human gene).

Hybridization for isolating a DNA encoding a functionally equivalentprotein can be carried out under the stringent conditions of, forexample, 10% formamide, 5×SSPE, 1×Denhardt's solution, and 1× salmonsperm DNA. More preferable (more stringent) conditions are, 25%formamide, 5×SSPE, 1×Denhardt's solution, and 1× salmon sperm DNA, andeven more preferable (even more stringent) conditions are, 50%formamide, 5×SSPE, 1×Denhardt's solution, and 1× salmon sperm DNA.However, several factors are thought to influence the stringency ofhybridization other than the above-described formamide concentration,and one skilled in the art can suitably select these factors toaccomplish a similar stringency. Also, instead of hybridization, it isalso possible to isolate a DNA encoding a functionally equivalentprotein by a gene amplification method such as PCR using a portion ofthe DNA encoding the protein (SEQ ID NO:1) as a primer.

The proteins encoded by DNA isolated by hybridization or gene amplifyingtechniques having functions equivalent to the protein of SEQ ID NO:2 areusually highly homologous to the protein of SEQ ID NO:2 at the aminoacid sequence level. The protein of the present invention also includesa protein that is functionally equivalent to the protein of SEQ ID NO:2and has a high homology to the amino acid sequence indicated in SEQ IDNO:2. “High homology” refers to an amino acid sequence identity of 40%or more, preferably 50% or more, and more preferably 60% or more. Thealgorithm described in the literature (Wilbur et al., Proc. Natl. Acad.Sci. USA 80:726-730, 1983) may be used for determining protein homology.

The protein of the present invention may be different in the amino acidsequence, molecular weight, isoelectric point, presence or absence of asugar chain, or the form of the sugar chain, and so forth depending onthe cells that produce it, the host, or purification process (describedlater). However, such proteins are included in the present inventionprovided the resulting protein is functionally equivalent to the proteindescribed in SEQ ID NO:2. For example, when the protein of the presentinvention is expressed in prokaryotic cells, for example, E. Coli, amethionine residue is added to the N-terminus of the amino acid sequenceof the original protein. Alternatively, when expressed in eukaryoticcells, for example, mammalian cells, the N-terminus signal sequence isremoved. The protein of the present invention also includes suchproteins. As a result of analyzing the amino acid sequence of theprotein of the present invention, the signal sequence was estimated toextend from Met at position 1 to Ser at position 24 in the amino acidsequence of SEQ ID NO:2. Thus, the present invention includes proteinscomprising amino acids from Cys at position 25 to Phe at position 222 inthe amino acid sequence described in SEQ ID NO:2.

The protein of the present invention can be prepared as a recombinantprotein or as a naturally-occurring protein by methods known to thoseskilled in the art. If it is a recombinant protein, the protein issecreted extracellularly as, for example, a soluble protein.Subsequently, the culture supernatant of the cells can be recovered,concentrated and then purified by chromatography utilizing ion exchange,reverse phase, or gel filtration chromatography, or by affinitychromatography using a column in which an antibody against the proteinof the present invention is immobilized, or by a combination of thesecolumns. Alternatively, the protein of the invention can be prepared byexpressing the protein in host cells (e.g., animal cells or E. coli) asa fusion protein with glutathione S transferase protein, or arecombinant protein with multiple histidine residues. The expressedprotein can be purified using a glutathione column or nickel column.Subsequently, if necessary, regions of the fusion protein (apart fromthe desired protein) can be digested and removed with thrombin or factorXa, etc. The natural form of the protein of the invention can beisolated by, for example, purifying a cell extract containing theprotein with an affinity column to which the antibody of the presentinvention described below is bound.

The present invention also includes partial peptides of the protein ofthe present invention. A partial peptide of the present inventioncomprises an amino acid sequence of at least seven amino acids,preferably eight or more amino acids, and more preferably nine or moreamino acids. The partial peptide can be used for, for example,production of an antibody against the protein of the present invention,screening of compounds that bind to the present protein, screening ofreceptors of the present protein, or preparation of a competitioninhibitor of the present protein. In addition, present inventionincludes partial peptides having, for example, the ability to bind to areceptor, but not the ability to activate the receptor (functioning as acompetitive inhibitor of the protein of the present invention). Partialpolypeptides of the present invention can be produced by geneticengineering techniques, known peptide synthesis methods, or by cleavingthe protein of the present invention with a suitable peptidase.

Moreover, the present invention relates to DNA encoding the protein ofthe present invention. In addition to being used for the production ofthe protein of the present invention either in vivo or in vitro aspreviously mentioned, the DNA of the present invention may also beapplied in, for example, gene therapy against diseases caused byaberrations of the gene encoding the protein of the present invention.Any type of DNA, such as cDNA synthesized from mRNA, genomic DNA, orsynthetic DNA, can be used, so long as the DNA encodes the protein ofthe present invention. Also as long as they can encode the presentprotein, DNAs comprising arbitrary sequences based on the degeneracy ofthe genetic code are also included.

The DNA of the present invention can be prepared by using methods knownin the art. For example, a cDNA library can be constructed from cellsexpressing the protein of the present invention and hybridization can beconducted using a part of the DNA sequence of the present invention (forexample, DNA sequence shown in SEQ ID NO:1) as a probe. Alternatively,the DNA of the present invention can be obtained by preparing RNA fromcells expressing the protein of the present invention, synthesizingoligo-DNAs based on the DNA sequence of the present invention (forexample, the DNA sequence shown in SEQ ID NO:1), and amplifying the cDNAencoding the protein of the present invention by PCR using theoligonucleotides as primers.

The nucleotide sequence of the obtained cDNA is determined to find anopen reading frame, and thereby the amino acid sequence of the proteinof the invention can be obtained. The cDNA obtained may also be used asa probe for screening a genomic library to isolate genomic DNA.

More specifically, mRNAs may first be prepared from a cell, tissue, ororgan (e.g., organs such as the lungs, liver, kidney, etc. or from anembryo) in which the protein of the invention is expressed. Knownmethods can be used to isolate mRNAs; for instance, total RNA isprepared by guanidine ultracentrifugation (Chirgwin et al., Biochemistry18:5294-5299, 1979) or AGPC method (Chomczynski et al., Anal. Biochem.162:156-159, 1987), and mRNA is purified from total RNA using an mRNAPurification Kit (Pharmacia), and such. Alternatively, mRNA may bedirectly purified by the QUICKPREP™ mRNA Purification Kit (Pharmacia).

The obtained mRNA is used to synthesize cDNA using reversetranscriptase. cDNA may be synthesized by using a kit such as the AMVReverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).Alternatively, cDNA may be synthesized and amplified following the5′-RACE method (Frohman et al., Proc. Natl. Acad. Sci. USA 85:8998-9002,1988; Belyavsky et al., Nucleic Acids Res. 17:2919-2932, 1989), usingthe synthesized DNA as a primer, the 5′-AMPLI FINDER RACE™ Kit(Clontech), and polymerase chain reaction (PCR).

A desired DNA fragment is prepared from the PCR products and ligatedwith a vector DNA. The recombinant vectors are used to transform E. coliand such, and a desired recombinant vector is prepared from a selectedcolony. The nucleotide sequence of the desired DNA may be verified byconventional methods, such as dideoxynucleotide chain termination.

The DNA of the invention may be designed to have a nucleotide sequencehaving a high expression efficiency by taking into account the frequencyof codon usage in the host used for the expression (Grantham et al.,Nucleic Acids Res. 9:43-74, 1981). The DNA of the present invention maybe altered by a commercially available kit or a conventional method. Forinstance, the DNA may be altered by digestion with restriction enzymes,insertion of a synthetic oligonucleotide or an appropriate DNA fragment,addition of a linker, or insertion of an initiation codon (ATG) and/orstop codon (TAA, TGA, or TAG).

Specifically, the DNA of the present invention includes a DNA comprisingnucleotides from nucleotide A at position 87 to nucleotide T at position752 of SEQ ID NO:1, and a DNA comprising nucleotides from nucleotide Tat position 159 to nucleotide T at position 752 in the nucleotidesequence of SEQ ID NO:1.

The DNA of the present invention also includes a DNA that hybridizes toa DNA comprising the nucleotide sequence indicated in SEQ ID NO:1 understringent conditions and is functionally equivalent to the proteindescribed in SEQ ID NO:2. Examples of hybridization conditions includethe conditions previously described. The hybridized DNA may preferablybe naturally occurring DNA, such as cDNA or chromosomal DNA.

The DNA of the present invention can be used to produce the protein ofthe present invention as a recombinant protein. In addition, when thereis a defect in the DNA encoding the protein of the present invention,the DNA of the present invention may also be applied to functionalinhibition by an antisense, or gene therapy by substituting it with thenormal gene.

As used herein, an “isolated nucleic acid” is a nucleic acid, thestructure of which is not identical to that of any naturally occurringnucleic acid or to that of any fragment of a naturally occurring genomicnucleic acid spanning more than three genes. The term therefore covers,for example, (a) a DNA which has the sequence of part of a naturallyoccurring genomic DNA molecule but is not flanked by both of the codingsequences that flank that part of the molecule in the genome of theorganism in which it naturally occurs; (b) a nucleic acid incorporatedinto a vector or into the genomic DNA of a prokaryote or eukaryote in amanner such that the resulting molecule is not identical to anynaturally occurring vector or genomic DNA; (c) a separate molecule suchas a cDNA, a genomic fragment, a fragment produced by polymerase chainreaction (PCR), or a restriction fragment; and (d) a recombinantnucleotide sequence that is part of a hybrid gene, i.e., a gene encodinga fusion protein. Specifically excluded from this definition are nucleicacids present in random, uncharacterized mixtures of different DNAmolecules, transfected cells, or cell clones, e.g., as these occur in aDNA library such as a cDNA or genomic DNA library.

Accordingly, in one aspect, the invention provides an isolated orpurified nucleic acid molecule that encodes a polypeptide describedherein or a fragment thereof. Preferably, the isolated nucleic acidmolecule includes a nucleotide sequence that is at least 60% identicalto the nucleotide sequence shown in SEQ ID NO:1. More preferably, theisolated nucleic acid molecule is at least 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, identical to thenucleotide sequence shown in SEQ ID NO:1. In the case of an isolatednucleic acid molecule which is longer than or equivalent in length tothe reference sequence, e.g., SEQ ID NO:1, the comparison is made withthe full length of the reference sequence. Where the isolated nucleicacid molecule is shorter that the reference sequence, e.g., shorter thanSEQ ID NO:1, the comparison is made to a segment of the referencesequence of the same length (excluding any loop required by the homologycalculation).

As used herein, “% identity” of two amino acid sequences, or of twonucleic acid sequences, is determined using the algorithm of Karlin andAltschul (PNAS USA 87:2264-2268, 1990), modified as in Karlin andAltschul, PNAS USA 90:5873-5877, 1993). Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul et al. (J.Mol. Biol. 215:403-410, 1990). BLAST nucleotide searches are performedwith the NBLAST program, score=100, wordlength=12. BLAST proteinsearches are performed with the XBLAST program, score=50, wordlength=3.To obtain gapped alignment for comparison purposes, GappedBLAST isutilized as described in Altschul et al (Nucleic Acids Res.25:3389-3402, 1997). When utilizing BLAST and GappedBLAST programs thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)are used to obtain nucleotide sequences homologous to a nucleic acidmolecule of the invention.

The present invention also relates to a vector into which the DNA of thepresent invention is inserted. The vectors of the present invention areuseful for carrying the DNA of the present invention and to express theprotein of the present invention in a host cell.

When E. coli is used as the host cell, any vector can be used as long asit comprises an “ori”, to amplify and mass-produce the vector in E. coli(e.g., JM109, DH5α, HB101, or XL1 Blue), and a marker gene for selectingthe transformed E. coli (e.g., a drug-resistant gene selected by a drug(e.g., ampicillin, tetracycline, kanamycin, or chloramphenicol). Forexample, M13-series vectors, pUC-series vectors, pBR322, pBluescript,pCR-Script, and such, can be used. Other than the vectors used above,pGEM-T, pDIRECT, pT7, and so on, can also be used for subcloning andexcision of the cDNA. When using a vector to produce the protein of thepresent invention, an expression vector is especially useful. When, forexample, the objective is to be expressed in E. coli, the expressionvector should have the above characteristics in order to be amplified inE. coli. When E. coli, such as JM109, DH5a, HB101, or XL1 Blue, are usedas the host cell, the vector should have a promoter, for example, lacZpromoter (Ward et al., Nature 341:544-546, 1989; FASEB J. 6:2422-2427,1992), araB promoter (Better et al., Science 240:1041-1043, 1988), or T7promoter, that can efficiently promote the expression of the desiredgene in E. coli. Other examples of the vectors are pGEX-5X-1(Pharmacia), QIAEXPRESS® system (Qiagen), pEGFP, and pET (for thisvector, BL21, a strain expressing T7 RNA polymerase, is preferably usedas the host).

In addition, a signal sequence for secreting a polypeptide may becontained in the vector. The pelB signal sequence (Lei et al., J.Bacteriol. 169:4379, 1987) can be used as the signal sequence forprotein secretion when the secretory protein is produced into theperiplasm of E. Coli. Introduction of the vector into host cells can becarried out using, for example, the calcium chloride method,electroporation, and so forth.

Examples of vectors used for producing the protein of the inventionother than those derived from E. Coli include, for example, expressionvectors derived from mammals (e.g., pcDNA3 (Invitrogen), pEGF-BOS(Nucleic Acids. Res. 18(17):5322, 1990), pEF, and pCDM8), those derivedfrom insect cells (e.g., the “Bac-to-BAC baculovirus expression system”(GIBCO BRL) and pBacPAK8), those derived from plants (e.g., pMH1 andpMH2), those derived from animal viruses (e.g., pHSV, pMV, andpAdexLcw), those derived from retroviruses (e.g., pZIpneo), thosederived from yeast (e.g., “Pichia Expression Kit” (Invitrogen), pNV11,and SP-Q01), and those derived from Bacillus subtilis (e.g., pPL608 andpKTH50).

For expression in animal cells such as CHO cells, COS cells, or NIH3T3cells, it is essential for the vector to have a promoter necessary forexpression in the cells, such as the SV40 promoter (Mulligan et al.,Nature 277:108, 1979), MMLV-LTR promoter, EF1α promoter (Mizushima etal., Nucleic Acids Res. 18:5322, 1990), and CMV promoter. Morepreferably, such a vector comprises a gene for selecting celltransformation (for example, a drug-resistant gene that allows selectionby a drug (such as neomycin or G418). Examples of vectors having suchcharacteristics include, for example, pMAM, pDR2, pBK-RSV, PBK-CMV,pOPRSV, and pOP13.

Moreover, for stably expressing a gene and amplifying the number ofcopies of the gene within cells, one example of a method that can beused is the method in which a vector (such as pCH01) having a DHFR genethat compensates for a defect in the nucleic acid synthesis pathway isinserted into CHO cells, which is followed by amplification bymethotrexate (MTX). For transient gene expression, an example method isone in which COS cells, having a gene on their chromosomes thatexpresses SV40 T antigen, are transformed with a vector (such as pcD)having an SV40 replication origin. Replication origins derived frompolioma virus, adenovirus, bovine papillomavirus (BPV), and so forth canbe also used. Moreover, in order to amplify the number of gene copies ina host cell line, the expression vector can contain aminoglycosidetransferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthineguanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolic acidreductase (dhfr) gene, and so forth, as a selective marker.

On the other hand, for expressing the DNA of the present invention inthe living body of animals, a method in which the DNA is firstincorporated into a suitable vector, and then the vector is introducedinto the living body by the retrovirus method, liposome method, cationicliposome method, adenovirus method, and so forth, may be used. Thereby,gene therapy can be carried out against diseases caused by a mutation inthe gene that encodes the protein of the present invention. For example,without limitation, adenovirus vectors (e.g., pAdexlcw) and retrovirusvectors (e.g., pZIPneo) are used as vectors. General gene manipulationtechniques such as the insertion of the DNA of the present inventioninto a vector can be carried out in accordance with ordinary methods(Molecular Cloning, 5.61-5.63). Administration into the living body maybe carried out by either the ex vivo method or in vivo method.

Furthermore, the present invention relates to a host cell into which thevector of the present invention has been introduced. There are noparticular restrictions on the host cell, and includes, for example, E.coli and various animal cells. The host cell of the present inventioncan be used, for example, as a production system for the production andexpression of the protein of the present invention. Production systemsfor producing the protein include both in vitro and in vivo systems.Examples of in vitro production systems include those using eukaryoticcells or prokaryotic cells.

When using eukaryotic cells, for example, animal cells, plant cells, andfungal cells can be used as the host. Known examples of animal cellsinclude mammalian cells such as CHO (J. Exp. Med. 108:945, 1995), COS,3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero, amphibian cellssuch as Xenopus oocytes (Valle et al., Nature 291:358-340, 1981), andinsect cells such as sf9, sf21, and Tn5. Particularly preferable CHOcells are those deficient in the DHFR gene, dhfr-CHO (Proc. Natl. Acad.Sci. USA 77:4216-4220, 1980) and CHO K-1 (Proc. Natl. Acad. Sci. USA60:1275, 1968). For mass expression in animal cells, CHO cells areparticularly preferable. A vector can be introduced into host cells by,for example, the calcium phosphate method, DEAE dextran method, methodusing cationic ribosome DOTAP (Boehringer-Mannheim), electroporation,and lipofection.

Known examples of plant cells used as protein production systems includecells derived from Nicotiana tabacum, and these cells can be cultured asa callus culture. Yeasts such as Saccharomyces species, e.g.,Saccharomyces cerevisiae, as well as filamentous bacteria such asAspergillus species, e.g., Aspergillus niger are known as fungal cells.

For prokaryotic cells, bacterial cells can be used as the productionsystem. Examples of bacterial cells include E. coli such as E. coliJM109, DH5a and HB101, as well as Bacillus subtilis.

These cells are transformed by desired DNA, and the resultingtransformants are cultured in vitro to obtain the protein. Transformantscan be cultured using known methods. Culture medium such as DMEM, MEM,RPMI1640, or IMDM may be used for animal cells with or without serumsupplements such as fetal calf serum (FCS). The pH of the culture mediumis preferably between about 6 and 8. Such cells are typically culturedat about 30 to 40° C. for about 15 to 200 hr, and the culture medium maybe replaced, aerated, or stirred as necessary.

Animal and plant hosts may be used for in vivo protein-producingsystems. For example, a desired DNA can be introduced into an animal orplant host. Encoded proteins are produced in vivo, and then recovered.These animal and plant hosts are included in the host cells of thepresent invention.

Animals to be used for the production systems described above include,but are not limited to, mammals and insects. Mammals such as goats,pigs, sheep, mice, and cattle may be used (Vicki Glaser, SPECTRUMBiotechnology Applications, 1993). Alternatively, the mammals may betransgenic animals.

For instance, a desired DNA may be prepared as a fusion gene by fusingit with a gene that encodes a protein specifically produced into milk,such as goat β casein gene. DNA fragments comprising the fusion gene areinjected into goat embryos, which are then introduced back into femalegoats. Proteins of interest can be recovered from milk produced by thetransgenic goats (i.e., those born from the goats that had received themodified embryos) or from their offspring. To increase the amount ofmilk containing the proteins produced by transgenic goats, appropriatehormones may be administered (Ebert et al., Bio/Technology 12:699-702,1994).

Alternatively, insects, such as the silkworm, may be used. A DNAencoding the desired protein inserted into a baculovirus can be used toinfect silkworms, and the desired protein can be recovered from theirbody fluids (Susumu et al., Nature 315:592-594, 1985).

As plants, for example, tobacco can be used. When using tobacco, DNAencoding the desired protein may be inserted into a plant expressionvector, such as pMON530, which is introduced into bacteria, such asAgrobacterium tumefaciens. Then the bacteria is used to infect tobacco,such as Nicotiana tabacum, and a desired polypeptide is recovered fromtheir leaves (Julian et al., Eur. J. Immunol. 24:131-138, 1994).

A protein of the present invention obtained as above may be isolatedfrom the inside or outside (such as the culture medium) of the hostcell, and purified as a substantially pure homogeneous protein. Themethod for protein isolation and purification is not limited to anyspecific method; in fact, any standard method may be used. For instance,column chromatography, filters, ultrafiltration, salt precipitation,solvent precipitation, solvent extraction, distillation,immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectricpoint electrophoresis, dialysis, and recrystallization may beappropriately selected and combined to isolate and purify the protein.

For chromatography, for example, affinity chromatography, ion-exchangechromatography, hydrophobic chromatography, gel filtration, reversephase chromatography, adsorption chromatography, and such may be used(Strategies for Protein Purification and Characterization: A LaboratoryCourse Manual. Ed. Daniel R. Marshak et al., Cold Spring HarborLaboratory Press, 1996). These chromatographies may be performed byliquid chromatography such as HPLC and FPLC. Thus, the present inventionprovides highly purified proteins produced by the above methods.

A protein of the present invention may be arbitrarily modified orpeptides may be partially deleted by treating it with an appropriateprotein modification enzyme before or after purification. Useful proteinmodification enzymes include, but are not limited to, trypsin,chymotrypsin, lysylendopeptidase, protein kinase, glucosidase and such.

The present invention also relates to an antibody that binds to theprotein of the invention. An antibody of the invention may take anyform, including monoclonal antibodies, as well as polyclonal antibodies.Furthermore, antiserum obtained by immunizing an animal such as arabbit, or the like, with the protein of the invention, all classes ofpolyclonal and monoclonal antibodies, human antibodies, and humanizedantibodies produced by genetic recombination are included.

A protein of the invention used as an antigen to obtain an antibody maybe derived from any animal species, but preferably it is from a mammalsuch as a human, mouse, or rat, more preferably from a human. Ahuman-derived protein may be obtained from the nucleotide or amino acidsequences disclosed herein.

A whole protein or a partial peptide of a protein may be used as asensitizing antigen in the present invention. A partial peptide may be,for example, an amino (N)-terminal or carboxy (C)-terminal fragment of aprotein. Herein, an “antibody” is defined as an antibody thatspecifically reacts with either the full length or a fragment of aprotein.

A gene encoding the protein of the invention or a its fragment may beinserted into a known expression vector, a host cell as described hereinmay be transformed with the vector, and the desired protein or itsfragment for use as a sensitizing antigen may be obtained from theoutside or inside of host cells by any standard method. Alternatively,cells expressing the protein or their lysates, or a chemicallysynthesized protein may be used as the antigen.

Any mammal may be immunized with the antigen, but preferably thecompatibility with parental cells used for cell fusion is taken intoaccount. In general, animals of Rodentia, Lagomorpha, or Primates areused.

Animals of Rodentia include, for example, mice, rats, and hamsters.Animals of Lagomorpha include, for example, rabbits. Animals of Primatesinclude, for example, monkeys of Catarrhini (old world monkey) such asMacaca fascicularis, rhesus monkeys, sacred baboons, or chimpanzees.

Methods for immunizing animals with antigens are known in the art. In astandard method, a sensitizing antigen is injected intraperitoneally orsubcutaneously to mammals. More specifically, an appropriate amount of astandard adjuvant, such as Freund's complete adjuvant, is mixed with thesensitizing antigen, diluted and suspended in an appropriate amount ofphosphate buffered saline (PBS), physiological saline, or such,emulsified, and then administered to mammals. Preferably, this isfollowed by several administrations of antigen mixed with anappropriately amount of Freund's incomplete adjuvant every 4 to 21 days.An appropriate carrier may also be used for immunization of sensitizingantigens. After an immunization as above, the serum is examined for anincrease of the amount of desired antibodies by a standard method.

Polyclonal antibodies against the protein of the present invention maybe prepared by collecting blood from the immunized mammal examined foran increase of desired antibodies in the serum, and by separating serumfrom the blood by any conventional method. Polyclonal antibodies may beused as serum containing polyclonal antibodies, or if necessary, afraction containing the polyclonal antibodies may be isolated from theserum for use. For example, immunoglobulin G or M can be prepared byusing an affinity column coupled with the protein of the presentinvention to obtain a fraction that recognizes only the protein,followed by purifying this fraction using a protein A column or proteinG column.

To prepare a monoclonal antibody, immune cells are collected from themammal sensitized with the above antigen after verifying that thedesired antibody level has increased in the serum. The cells are thensubjected to cell fusion. The immune cells used for cell fusion arepreferably obtained from spleen. The other parent cell that is fusedwith the above immune cell is preferably a mammalian myeloma cell, andmore preferably a myeloma cell that has acquired a special feature thatcan be used for the selection of fusion cells by a drug.

Cell fusion of the above immune cell and myeloma cell may be performedby any standard method, such as those described in literature (Galfre etal., Methods Enzymol. 73:3-46, 1981).

Resulting hybridomas obtained by cell fusion may be selected bycultivating them in a standard selection medium, such as HAT medium(hypoxanthine, aminopterin, and thymidine containing medium). The cellculture is typically continued in the HAT medium until all cells otherthan the desired hybridoma (non-fused cells) die, usually from severaldays to several weeks. Then, the standard limiting dilution is performedto screen and clone a hybridoma cell producing the desired antibody.

Besides the above method in which a nonhuman animal is immunized with anantigen for preparing hybridoma, human lymphocytes such as thoseinfected by the EB virus may be immunized with a protein, proteinexpressing cells, or their lysates in vitro. Then, the immunizedlymphocytes are fused with human-derived myeloma cells capable ofindefinite division, such as U266, to yield a hybridoma producing adesired human antibody capable of binding to the protein (UnexaminedPublished Japanese Patent Application (JP-A) No. Sho 63-17688).

Subsequently, the hybridomas thus obtained are transplanted into theabdominal cavity of a mouse from which the ascites is collected. Themonoclonal antibodies thus obtained can be purified by, for example,ammonium sulfate precipitation or by column chromatography using aprotein A or protein G column, a DEAE ion exchange column, an affinitycolumn, and such to which the protein of the invention is coupled. Theantibody of the invention can be used not only for purifying anddetecting a protein of the invention, but also as a candidate for anagonist or antagonist to a protein of the present invention. Theantibody is also expected to be used in antibody therapy againstdiseases related to the present protein. When using the resultingantibody for the purpose of administration to the human body (antibodytherapy), human antibodies or humanized antibodies are preferred toreduce immunogenicity.

For example, human antibodies against a protein can be obtained usinghybridomas obtained by fusing myelomas with antibody-producing cells,which are obtained by immunizing transgenic animals having the humanantibody gene repertoire with an antigenic protein, cells expressing theprotein, or a lysate thereof (See WO92-03918, WO93-2227, WO94-02602,WO94-25585, WO96-33735 and WO96-34096).

Alternatively, an immune cell, such as an immunized lymphocyte, whichproduces antibodies may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

Monoclonal antibodies thus obtained can be also be recombinantlyprepared using conventional genetic engineering techniques (see, forexample, Borrebaeck C. A. K. and Larrick J. W. Therapeutic MonoclonalAntibodies, published in the United Kingdom by MacMillan Publishers LTD,1990). A recombinant antibody can be produced by cloning a DNA encodingthe antibody from an immune cell such as a hybridoma or an immunizedlymphocyte producing the antibody, inserting this DNA into anappropriate vector, and introducing this into a host cell. The presentinvention also provides recombinant antibodies prepared as describedabove.

An antibody of the present invention may be a fragment of an antibody ora modified antibody, so long as it binds to one or more of the proteinsof the invention. For instance, the antibody fragment may be Fab, F(ab′)₂, Fv, or single chain Fv (scFv), in which Fv fragments from H andL chains are ligated by an appropriate linker (Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883, 1988). More specifically, an antibodyfragment may be generated by treating an antibody with an enzyme such aspapain or pepsin. Alternatively, a gene encoding the antibody fragmentmay be constructed, inserted into an expression vector, and expressed inan appropriate host cell (see, for example, Co et al., J. Immunol.152:2968-2976, 1994; Better et al., Methods Enzymol. 178:476-496, 1989;Pluckthun et al., Methods Enzymol. 178:497-515, 1989; Lamoyi, MethodsEnzymol. 121:652-663, 1986; Rousseaux et al., Methods Enzymol.121:663-669, 1986; Bird et al., Trends Biotechnol. 9:132-137, 1991).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention encompassessuch modified antibodies. A modified antibody can be obtained bychemically modifying an antibody. These modification methods areconventional in the field.

An antibody of the present invention may be obtained as a chimericantibody comprising a variable region derived from a nonhuman antibodyand the constant region derived from a human antibody. Alternatively,the present antibody may be obtained as a humanized antibody, comprisingthe complementarity-determining region (CDR) derived from a nonhumanantibody, the framework region (FR) constant region derived from a humanantibody. Such antibodies can be prepared by using known techniques.

An Antibody obtained as above may be purified to homogeneity. Methodsgenerally used for separating and purifying ordinary proteins may beused for separating and purifying the present antibody. For example, theantibody can be separated and/or purified by the appropriate selectionand combined use of column chromatographies such as affinitychromatography and the like, filters, ultrafiltration, salting-out,dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing,etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, ColdSpring Harbor Laboratory, 1988), without limitation. The concentrationof the antibody obtained as above may be determined by measuring theabsorbance or by an enzyme-linked immunosorbent assay (ELISA), and such.

A column used in affinity chromatography is exemplified by protein Acolumn or protein G column. For example, protein A column includes HYPERD™, POROS™, and SEPHAROSE™ F. F. (Pharmacia).

In addition to affinity chromatography, the chromatography methodincludes, for example, ion-exchange chromatography, hydrophobicchromatography, gel filtration, reverse-phase chromatography, adsorptionchromatography, and the like (Strategies for Protein Purification andCharacterization: A Laboratory Course Manual. Ed Daniel R. Marshak etal., Cold Spring Harbor Laboratory Press, 1996). The chromatographiescan be carried out by liquid-phase chromatography such as HPLC, FPLC, orthe like.

For example, the determination of absorbance, Enzyme-linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay(RIA), and/or immunofluorescence may be used to measure the antigenbinding activity of the antibody of the invention. In ELISA, theantibody of the present invention is immobilized on a plate, protein ofthe invention is applied to the plate, and then a sample containing adesired antibody, such as culture supernatant of antibody producingcells or purified antibodies, is applied. Then, a secondary antibody,which recognizes the primary antibody and which is labeled with anenzyme such as alkaline phosphatase, is applied, and the plate isincubated. After washing, an enzyme substrate, such as p-nitrophenylphosphate, is added to the plate, and the absorbance is measured toevaluate the antigen binding activity of the sample. A fragment of theprotein, such as a C-terminal or N-terminal fragment, may also be used.BIACORE® (Pharmacia) may be used to evaluate the activity of theantibody according to the present invention.

The above methods allow the detection or measurement of the protein ofthe invention, by exposing the antibody of the invention to a samplepresumed to contain the protein of the invention, and detecting ormeasuring the immune complex formed by the antibody and the protein.

Because the method of detection or measurement of the protein accordingto the invention can specifically detect or measure a protein, themethod may be useful in a variety of experiments in which the protein isused.

The present invention also relates to a nucleotide comprising at least15 nucleotides that hybridizes to the DNA (SEQ ID NO:1) encoding theprotein described in SEQ ID NO:2 or to a complementary strand thereof.Nucleotides of the present invention specifically hybridize to DNA (SEQID NO:1) encoding the protein described in SEQ ID NO:2 or to acomplementary strand thereof. Here, the term “specifically hybridize”refers to the absence of significant cross-hybridization with DNAencoding other proteins under normal hybridization conditions, andpreferably under stringent hybridization conditions. Such nucleotidesinclude probes, primers, nucleotides, and nucleotide derivatives (e.g.,DNA encoding antisense oligonucleotides and ribozyme) that are able tospecifically hybridize to DNA encoding the protein of the presentinvention or to complementary DNA thereof. In addition, such nucleotidescan also be used for the production of DNA chips.

The present invention comprises, for example, an antisenseoligonucleotide that hybridizes to any part of the nucleotide sequenceof SEQ ID NO:1. The antisense oligonucleotide is preferably an antisenseof a continuous sequence comprising at least 15 nucleotides or morewithin the nucleotide sequence SEQ ID NO:1. More preferably, the abovecontinuous sequence comprising at least 15 nucleotides or more thatcontains a translation initiation codon.

A derivative or modified form of an antisense oligonucleotide may alsobe used. The latter form may be modified with lower alkylphosphonatesuch as methylphosphonate or ethylphosphonate, or with phosphorothioate,or phosphoroamidate.

Herein, an antisense oligonucleotide is not restricted to one in whichall nucleotides are complementary to the corresponding nucleotideswithin a given region of a DNA or mRNA, as long as it can specificallyhybridize to the nucleotide sequence of SEQ ID NO:1, it may have one ormore nucleotide mismatches.

Such nucleotides have a homology of at least 70%, preferably 80% ormore, more preferably 90% or more, and even more preferably 95% or morewithin a continuous sequence comprising at least 15 nucleotides or more.To determine the degree of homology, the algorithm described herein maybe used. As described in the following Examples, the above DNA is usefulas a probe for detecting or isolating a DNA encoding the protein of theinvention, or as a primer for its amplification.

A derivative of an antisense oligonucleotide of a present invention mayact on cells producing the protein of the invention and bind to a DNA ormRNA encoding the protein, and then, it may inhibit the expression ofthe protein of the invention by inhibiting its transcription ortranslation, or by promoting the degradation of mRNA, and therebyinhibiting the function of the protein of the invention.

A derivative of an antisense oligonucleotide of the present inventionmay be mixed with an appropriate nucleotide that is inactive against thederivative, and used as a medicine for external application, such as anointment or poultice.

If necessary, it may be mixed with an excipient, isotonizing agent,solubilizing agent, stabilizer, preservative, pain-killer, or the like,and prepared as a tablet, powder, granule, capsule, liposome capsule,injectable solution, liquid formulation, nose drops, freeze-dried agent,etc. The above may be achieved according to standard methods.

For treating patients, a derivative of an antisense oligonucleotide ofthe present invention may be, for example, directly applied to theaffected area of a patient, or administered into blood vessels so as tofinally reach the affected area. Moreover, the derivative may beencapsulated in antisense-encapsulating materials such as liposomes,poly-L-lysine, lipid, cholesterol, lipofectin, or their derivatives inorder to increase durability and/or membrane permeability.

Dose of the derivative of the antisense oligonucleotide of the presentinvention may be appropriately adjusted depending on the patient'scondition, and an appropriate amount such as 0.1 to 100 mg/kg, or morepreferably 0.1 to 50 mg/kg may be administered.

As an antisense oligonucleotide of the present invention inhibitsexpression of the protein of the invention, it can be utilized as aninhibitor of a biological activity of the protein of the invention. Aninhibitor of expression comprising an antisense oligonucleotide of thepresent invention is useful because it can inhibit the a biologicalactivity of the protein of the invention.

Further, the present invention relates to a method for screeningcompounds that bind to the protein of the present invention using theprotein, as well as compounds which can be isolated by the screeningmethod (e.g., receptors, agonists, and antagonists).

The protein of the present invention used for screening may be arecombinant protein, natural protein, or a partial peptide. Oneembodiment of this screening method comprises the steps of (a)contacting a test sample with the protein of the present invention orits partial peptide, and (b) selecting a compound having an activity forbinding to the protein or its partial peptide. Without limitation, thetest sample includes cell extracts, cell culture supernatants, productsof fermentation microorganisms, marine organism extracts, plantextracts, purified or crude proteins, peptides, non-peptide compounds,synthetic low molecular weight compounds, and naturally-occurringcompounds. The protein of the present invention can be contacted withthe test sample in the form of, for example, a purified protein,solubilized protein, a protein bound to a carrier, a fusion protein withanother protein, a protein expressed on a cell membrane, or as amembrane fraction.

Numerous methods known to those skilled in the art can be used asmethods for screening a protein that binds to the protein of the presentinvention using the protein. Such screenings can be carried out, forexample, by the immunoprecipitation method. Specifically, the method canbe carried out as follows. The gene encoding the protein of thisinvention is expressed by inserting the gene downstream of a promoterfor expressing a foreign gene, such as pSV2neo, pcDNA I, pCD8, etc., andexpressing the gene in animal cells, etc. Any generally used promotermay be employed for the expression, including the SV40 early promoter(Rigby In Williamson (ed.), Genetic Engineering, vol. 3. Academic Press,London, p. 83-141, 1982), EF-1α promoter (Kim et al., Gene 91:217-223,1990), CAG promoter (Niwa et al., Gene 108:193-200, 1991), RSV LTRpromoter (Cullen, Methods in Enzymol. 152:684-704, 1987), SRα promoter(Takebe et al., Mol. Cell. Biol. 8:466, 1988), CMV immediate earlypromoter (Seed et al., Proc. Natl. Acad. Sci. USA 84:3365-3369, 1987),SV40 late promoter (Gheysen et al., J. Mol. Appl. Genet. 1:385-394,1982), Adenovirus late promoter (Kaufman et al., Mol. Cell. Biol. 9:946,1989), HSV TK promoter, etc. Transfer of a foreign gene into animalcells for expression can be performed by any one of the followingmethods, including the electroporation method (Chu et al., Nucl. AcidRes. 15:1311-1326, 1987), the calcium phosphate method (Chen et al.,Mol. Cell. Biol. 7:2745-2752, 1987), the DEAE dextran method (Lopata etal., Nucl. Acids Res. 12:5707-5717, 1984; Sussman et al., Mol. Cell.Biol. 4:1642-1643, 1985), the lipofectin method (Derijard, Cell7:1025-1037, 1994; Lamb et al., Nature Genetics 5:22-30, 1993; Rabindranet al., Science 259:230-234, 1993), etc. The protein of this inventioncan be expressed as a fusion protein having a recognition site for amonoclonal antibody by introducing such a site the specificity of whichhas been established, into the N- or C-terminal of the protein of thisinvention. For this purpose, a commercial epitope-antibody system can beutilized (Jikken Igaku, Exp. Med. 13:85-90, 1995). Vectors arecommercially available which are capable of expressing fusion proteinswith β-galactosidase, maltose-binding protein, glutathioneS-transferase, green fluorescence protein (GFP), and such, via amulti-cloning site.

To minimize the alteration in properties of the protein of thisinvention due to fusion protein formation, a method for preparing afusion protein by introducing only a small epitope portion comprisingseveral to ten amino acid residues has been reported. For example, theepitopes of polyhistidine (His-tag), influenza hemagglutinin (HA), humanc-myc, FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene10 protein (T7-tag), human herpes simplex virus glycoprotein (HSV-tag),E-tag (epitope on the monoclonal phage), and such, and monoclonalantibodies that recognize these epitopes can be utilized asepitope-antibody systems for screening proteins binding to the proteinof this invention (Jikken Igaku, Exp. Med. 13:85-90, 1995).

In immunoprecipitation, immune complexes are formed by adding theseantibodies to a cell lysate prepared using suitable surfactants. Thisimmune complex comprises the protein of this invention, a proteincapable of binding to the protein, and an antibody. Immunoprecipitationcan also be performed using an antibody against the protein of thisinvention besides antibodies to the above-described epitopes. Anantibody to the protein of this invention can be prepared by inserting agene encoding the protein of this invention into an appropriateexpression vector of E. coli to express it in the bacterium, purifyingthe protein thus expressed, and immunizing rabbits, mice, rats, goats,chicken, and such, with the purified protein. The antibody can also beprepared by immunizing the above-described animals with a partialpeptide of the protein of this invention.

Immune complexes can be precipitated using, for example, Protein ASepharose and Protein G Sepharose when the antibody is a murine IgGantibody. In addition, in the case where the protein of this inventionis prepared as a fusion protein with an epitope of, for example, GST,and such, the immune complex can be formed using a substance thatspecifically binds to this epitope, such as glutathione-Sepharose 4B,and such, giving the same result as in the case where the antibody forthe protein of this invention is used.

Immunoprecipitation, in general, may be carried out according to, orfollowing the method described in literature (Harlow et al.: Antibodies,pp. 511-552, Cold Spring Harbor Laboratory publications, New York,1988).

SDS-PAGE is generally used for the analysis of immunoprecipitatedproteins. Bound proteins can be analyzed based on the molecular weightsof proteins using a gel of an appropriate concentration. In this case,although proteins bound to the protein of this invention, in general,are hardly detectable by the usual protein staining methods, such asCoomassie staining and silver staining, the detection sensitivity can beimproved by culturing cells in a medium containing radio isotope-labeled³⁵S-methionine and ³⁵S-cysteine to label proteins inside the cells, anddetecting the labeled proteins. Once the molecular weight of the proteinis determined, the desired protein can be purified directly fromSDS-polyacrylamide gel and sequenced.

In addition, for example, West Western blotting method (Skolnik et al.,Cell 65:83-90, 1991) can be used to isolate a protein that binds to theprotein of the present invention using the protein. Namely, a cDNAlibrary is prepared using a phage vector (such as λgt11 or ZAP) fromcells, tissue, or organs (such as the heart, lung, liver, kidney, and soforth, and embryos) in which a protein that binds to the protein of thepresent invention is presumed to be expressed, and then, this cDNAlibrary is expressed on LB-agarose. The expressed protein is fixed on afilter and the fixed protein is purified, the labeled protein of thepresent invention is reacted with the above filter, and plagues thatexpress a protein bound to the protein of the present invention isdetected using the label. Methods for labeling a protein of the presentinvention include those that use the binding properties of biotin andavidin, those that use antibodies that specifically bind to the proteinof the present invention or a peptide or polypeptide (such as GST) thathas been fused with the protein, those that use radioisotopes orfluorescence.

In addition, other embodiments of the screening method of the presentinvention include methods that use a two-hybrid system using cells (suchas the “MATCHMAKER™ Two-Hybrid System”, “Mammalian MATCHMAKER™Two-Hybrid Assay Kit”, and “MATCHMAKER™ One-Hybrid System” (all byClontech), the “HYBRIZAP™ Two-Hybrid Vector System” (Stratagene), andthe “CYTO TRAP™ two-hybrid system” (Stratagene); References: Dalton etal., Cell 68:597-612, 1992; Fields et al., Trends. Genet. 10:286-292,1994).

In a two-hybrid system, a cDNA library is prepared from cells in whichprotein that bind to the protein of the present invention is presumed tobe expressed by expressing the protein of the present invention in yeastcells by fusing it with the SRF DNA binding region or GAL4 DNA bindingregion, and expressing in a form that fuses with the VP 16 or GAL4transcriptional activation region. This cDNA library is then introducedinto the above yeast cells, and library-derived cDNA is isolated fromthe positive clones detected (when a protein that binds to the proteinof the present invention is expressed in yeast cells, a reporter gene isactivated by their binding, thereby making it possible to confirm thepositive clones). Protein corresponding to the isolated cDNA can then beobtained by introducing said cDNA into E. coli and expressing ittherein.

Examples of reporter genes that can be used include HIS3 gene, Ade2gene, LacZ gene, CAT gene, luciferase gene and PAI-1 (plasminogenactivator inhibitor type 1) gene.

Screening of compounds that bind to the protein of the present inventioncan be carried out using affinity chromatography. For example, theprotein of the present invention is immobilized on a carrier of anaffinity column, and then a test sample presumed to express a proteinthat binds to the protein of the present invention is applied to thecolumn. In this case, for example, cell extracts and cell lysates can beused as test samples. After applying the test sample, the protein thatbinds to the protein of the present invention can be prepared by washingthe column.

DNA encoding the resulting protein can be obtained by analyzing theamino acid sequence of the protein, synthesizing oligo DNA based on thatsequence, and screening a cDNA library by using the DNA as a probe.

In the present invention, a biosensor utilizing the surface plasmonresonance phenomena can be used as means for detecting or determiningbound compounds. Biosensors using surface plasmon resonance phenomenaallow real-time observation of the interaction between the protein ofthe present invention and a test compound as a surface plasmon resonancesignal using a trace amount of the protein and without labeling (forexample, BIACORE® (Pharmacia)). Thus, using a BIACORE® or otherbiosensor allows one to evaluate the binding between the protein of thepresent invention and a test compound.

In addition, examples of methods known to persons skilled in the art forisolating compounds (including agonists and antagonists) that bind tothe protein of the present invention without being limited to proteins,include: a method for screening molecules that bind to the protein ofthe present invention by allowing a synthetic compound, naturalsubstance bank, or random phage peptide display library to act on animmobilized protein of the present invention; and a high-throughputscreening method using combinatorial chemistry technology (Wrighton etal., Science 273:458-464, 1996; Verdine, Nature 384:11-13, 1996; Hogan,Jr., Nature 384:17-19, 1996).

Compounds that can be isolated by the screening can be drug candidatesfor promoting or inhibiting the activity of the protein of the presentinvention, and can be applied to the treatment of diseases caused byexpression or functional abnormalities of the protein of the invention.A substance in which a portion of the structure of a compound, which hasan activity for binding to the protein of the present invention andwhich was obtained by using the screening method of the presentinvention, is altered by addition, deletion, and/or substitution is alsoincluded in the compounds obtained by using the screening method of thepresent invention.

When using the compound obtained by the screening method of thisinvention and present protein as a drug for humans and mammals, forexample, mice, rats, guinea pigs, rabbits, chicken, cats, dogs, sheep,pigs, cattle, monkeys, sacred baboons, and chimpanzees, the isolatedcompound itself can be directly administered to a patient, or it can begiven after formulation by using commonly known pharmaceuticalpreparation methods. For example, according to the need, the drug can betaken orally as sugarcoated tablets, capsules, elixirs, andmicrocapsules, or parenterally in the form of injections of asepticsolutions or suspensions with water or any other pharmaceuticallyacceptable liquid. The compound may be formulated by mixing with, forexample, pharmacologically acceptable carriers or media, specifically,sterilized water, physiological saline, plant oils, emulsifiers,suspending agents, surfactants, stabilizers, flavoring agents,excipients, vehicles, preservatives, binders, and so on, in a unit doseform required for generally accepted drug implementation. The amount ofactive ingredients in these preparations makes a suitable dosage withinthe indicated range acquirable.

Examples for additives which can be mixed with tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum, and arabic gum;excipients such as crystalline cellulose; swelling agents such as cornstarch, gelatin, and alginic acid; lubricators such as magnesiumstearate; sweeteners such as sucrose, lactose, or saccharin; andflavoring agents such as peppermint, Gaultheria adenothrix oil, andcherry. When the unit dosage form is a capsule, a liquid carrier, suchas oil, can also be included in the above ingredients. Sterilecompositions for injections can be formulated following normal drugimplementations using vehicles such as distilled water used forinjections.

Physiological saline and isotonic liquids including glucose or otheradjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol andpolyethylene glycol, non-ionic surfactants, such as polysorbate 80™ andHCO-50.

Sesame oil or soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizer; may be formulated with a buffer such as phosphate bufferand sodium acetate buffer; a pain-killer such as procaine hydrochloride;a stabilizer such as benzyl alcohol and phenol; or an anti-oxidant. Theprepared injection is filled into a suitable ampule.

The administration to patients is done by methods commonly known tothose skilled in the art, such as by intra-arterial, intravenous, orsubcutaneous injections, and in addition, as intranasal, bronchial,intramuscular, percutaneous, or oral administrations. One skilled in theart can suitably select the dosage according to the body-weight or ageof a patient, or the method of administration. Also, if the compound canbe encoded by DNA, the compound can be used for gene therapy byintegrating the DNA into a vector for gene therapy. Although the dosageamount and method of administration differ according to the body-weight,age, and symptoms of a patient, one skilled in the art can suitablyselect these.

For example, although the dosage for a single administration of theprotein of the present invention varies depending on the administrationtarget, target organ, symptoms, administration method, and so forth, thedosage in the form of, for example, an injection preparation is usuallyconsidered to be in the range of about 100 μg to 20 mg per day for anadult (assuming the body weight is 60 kg).

For example, although the dosage of a compound that binds to the proteinof the present invention or the dosage of a compound that inhibits theactivity of the protein of the present invention varies according to thesymptoms, in the case of oral administration, the dosage for an adult(assuming the body weight is 60 kg) is typically in the range of about0.1 to 100 mg, preferably about 1.0 to 50 mg, and more preferably about1.0 to 20 mg, per day.

In the case of parenteral administration, although the dosage for asingle administration also varies according to the administrationtarget, target organ, symptoms, and administration method, the dosage inthe form of, for example, an injection preparation for an adult(assuming the body weight is 60 kg) is usually in the range of about0.01 to 30 mg, preferably about 0.1 to 20 mg, and more preferably about0.1 to 10 mg, per day for a convenient intravenous administration. Inthe case of other animals as well, it is possible to administer anamount converted to 60 kg of body weight or per area of body surface.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows the homology between the amino acid sequences encoded byclone 106 (above; SEQ ID NO:2) and Drosophila twisted gastrulation (TSG)gene (below; SEQ ID NO:5). Asterisks (*) indicate identical amino acidsequences, while dots (.) indicate similar amino acid sequences. Gapsare supplemented with bars.

All publications and patents cited herein are incorporated by referencein their entirety.

DETAILED DESCRIPTION

The present invention is described below in detail using examples, butit is not to be construed as being limited thereto.

Example 1 Isolation of Clone 106

The AGM region was sampled from 11.5-day mice embryos, and polyA(+) RNAwas prepared using FAST TRACK® (Invitrogen). Double-strand cDNA wassynthesized using a random primer of the SUPERSCRIPT™ Choice System(GIBCO BRL). BstXI adapter (Invitrogen) was added after blunting theends of the cDNA, and then 400 bp or longer cDNA were fractionated usingthe SizeSep 400 Spun Column (Pharmacia).

After the cDNA was mixed with pMXGM(−)v-mpl^(M2) (see Japanese PatentApplication No. Hei 9-324912), which had been treated with BstX1(TAKARA) beforehand, it was ligated using T4 DNA ligase. The resultingDNA was introduced into E. coli DH10B (GIBCO BRL) by electroporationusing GENE PULSER® (BioRad), and cultured overnight. The cDNA librarywas purified using the JETSTAR™ column (GENOMED).

Packaging cells BOSC23 (Proc. Natl. Acad. Sci. USA 90:8392-8396, 1993)were transfected with the cDNA library using LIPOFECTAMINE™ (LIFETECHNOLOGIES). BOSC23 were seeded into a 6-cm dish with DMEM (LIFETECHNOLOGIES) containing 10% fetal calf serum (FCS, JRH BIOSCIENCES),and then washed with DMEM 16 hours later. 18 μl of LIPOFECTAMINE™diluted beforehand with 200 μl of DMEM and 3 μg of the cDNA librarydiluted with 200 μl of DMEM were mixed together. This was kept standingat room temperature for 15 minutes, then 1.6 ml of DMEM was addedthereto, and the mixture was added to the cells. After five hours, 2 mlof DMEM containing 20% FCS was added to the mixture and cultured for 19hours. Subsequently, the medium was replaced with 3 ml of DMEMcontaining 10% FCS and the culture supernatant was collected 24 hourslater. Mouse interleukin-3 (IL-3) and 10 μg/ml of hexadimethrine bromidewere added to the culture supernatant containing the recombinant virus,and Ba/F3 were suspended for infection. 24 hours after the infection,the cells were washed three times with PBS, and further cultured withRPMI1640 (LIFE TECHNOLOGIES) containing 10% FCS. DNA was extracted fromclones that proliferated in the absence of IL-3 and amplified by PCRusing primers 5′-gggggTggACCATCCTCTA-3′ (SEQ ID NO:3) and5′-CgCgCAgCTgTAAACggTAg-3′ (SEQ ID NO:4), designed to surround the cDNAinsertion site, followed by recovery of the cDNA fragment. PCR wasperformed under the following conditions with the GENEAMP® PCR System2400 (Applied Biosystems) using 50 μl of the reaction mixture containing500 ng of DNA, 500 μM each of primer, 2.5 units of TAKARA LA™ Taq(TAKARA), 2.5 mM MgCl₂, 0.3 mM dNTPs, and enzyme-supplemented buffer:denaturing at 98° C. for 60 seconds, followed by 30 cycles of 98° C. for20 seconds, and 68° C. for 120 seconds. The PCR reaction product waselectrophoresed on an agarose gel, the portion containing the amplifiedfragment was excised, and then purified. The nucleotide sequence of theresulting DNA fragment was determined and translated to amino acids,then the isolated gene (clone 106) was found to be 33% homologous at theamino acid level with the Drosophila twisted gastrulation gene (TSG)(Mason et al., Genes and Develop. 8:1489-1501) (FIG. 1). Drosophila TSGgene is thought to be one of the embryonic dorsal determining factors,and the mutation of this gene prevents differentiation of only dorsalmidline cells derived from the mesoderm. This is considerably differentto the decapentaplegic (DP) gene, which is also a dorsal determiningfactor considered to interact with TSG gene, where the differentiationof the entire dorsal region is affected.

Example 2 Acquisition of Full-Length cDNA

A cDNA library of a 11.5 day-mouse embryo was synthesized in the samemanner as in Example 1 using an oligo dT primer and screened using thecDNA fragment as the probe to obtain the full-length cDNA. 2 μg of thecDNA library was added to 50 μl of DH5α (GIBCO BRL) and left standingfor 30 minutes on ice. After applying heat shock for 30 seconds at 42°C., the mixture was allowed to stand for about 2 minutes on ice. Afterthe addition of 300 μl of SOC, the mixture was cultured for 30 minutesat 37° C. The mixture was then seeded into a 10-cm dish LB plate(containing ampicillin) on which a NITROBIND™ Nitrocellulose Transfermembrane (MICRON SEPARATIONS) was placed so as to obtain 30,000-40,000E. coli colonies per plate. The E. Coli colonies that proliferated onthe membrane were transferred to a BIODYNE® A transfer membrane (Pall),and cultivated on the LB plate for several hours. The BIODYNE® Atransfer membrane was then used for screening the cDNA library. Afterdenaturing with a denaturing solution (0.5 N NaOH and 0.5 M NaCl) forfive minutes, the membrane was neutralized with a neutralizing solution(0.5 M Tris-HCl, pH 7.4 and 1.5 M NaCl). After gently washing with 2×SSCand drying up, the DNA and membrane were cross-linked by irradiatingwith UV light at 1200 J.

Hybridization was performed according to the following procedure. First,the membrane was pre-hybridized for 2 hours at 42° C. in a hybridizationbuffer (50% formamide, 4.5% Dextran Sulfate, 0.1 mg/ml of salmon spermDNA, 6×SSC, and 1% SDS). After labeling with RI using PRIME-IT®(Stratagene) and after heat denaturing, 25 ng of clone 106 DNA to beused for the probe was added to the hybridization buffer and left tostand overnight.

The membrane was washed in two stages. First, the membrane was washedfor 10 minutes at 42° C. with a washing buffer (2×SSC and 0.1% SDS), andthen for 30 minutes at 55° C. with a washing buffer (0.1×SSC and 0.1%SDS). The membrane washed in this manner was then brought into closecontact with an X-ray film and developed by exposing to light at −80° C.

One type of clone was obtained through the above procedure. The clone,which was a 3986 bp cDNA, was found to have an open reading frame(87-752) that encodes 222 amino acids, in which amino acids 1 through 24were presumed to be the signal sequence. The nucleotide sequence of thecDNA is shown in SEQ ID NO:1, while the encoded amino acid sequence isshown in SEQ ID NO:2.

Example 3 Expression Analysis of the cDNA Clone by NorthernHybridization

When Northern hybridization was performed using mouse Multiple TissueNorthern Blot (Clontech) and the cDNA obtained in Example 2 as theprobe, signals of about 4.0 kb were found in the heart, lung, liver, andkidney. These signals were also confirmed to be expressed in 9, 10, 11,12, and 13-day embryos.

INDUSTRIAL APPLICABILITY

The protein and gene discovered in the present invention could becounterparts of Drosophila TSG gene in mice, which suggests that theymay be functionally similar. Through the investigation of their roles inembryo development, the protein and gene of the present invention maycontribute to the elucidation of mechanisms of differentiation and boneformation associated with hematopoietic stem cell generation. Inaddition, they are also useful as tools for developing therapeuticagents for the treatment of diseases related to immune andhematopoiesis-systems and bone formation.

1. A substantially purified polypeptide comprising residues 25-222 ofSEQ ID NO:2.
 2. The polypeptide of claim 1, wherein the polypeptidefurther comprises an initiator methionine or a signal peptide.
 3. Thepolypeptide of claim 1, wherein the polypeptide consists of residues25-222 of SEQ ID NO:2 plus an initiator methionine or a signal peptide.4. The polypeptide of claim 1, wherein the polypeptide comprises theamino acid sequence of SEQ ID NO:2.
 5. The polypeptide of claim 1,wherein the polypeptide consists of the amino acid sequence of SEQ IDNO:2.
 6. The polypeptide of claim 1, wherein the polypeptide comprisesthe amino acid sequence of SEQ ID NO:2 fused to a second amino acidsequence.
 7. The polypeptide of claim 1, wherein the polypeptidecomprises the amino acid sequence of residues 25-222 of SEQ ID NO:2fused to a second amino acid sequence.
 8. The polypeptide of claim 7,wherein the second amino acid sequence is a peptide selected from thegroup consisting of glutathione S-transferase, FLAG, six histidineresidues, influenza agglutinin (HA), human c-myc fragment, VSV-GPfragment, p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigenfragment, lck tag, α-tubulin fragment, B-tag, Protein C fragment,immunoglobulin constant region, β-galactosidase, Green FluorescentProtein (GFP), and maltose binding protein.
 9. The polypeptide of claim7, wherein the polypeptide comprises an initiator methionine.
 10. Thepolypeptide of claim 7, wherein the polypeptide further comprisesresidues 1-24 of SEQ ID NO:2.
 11. The polypeptide of claim 7, whereinthe polypeptide comprises a signal sequence.
 12. A substantiallypurified polypeptide that (a) has at least 95% identity to the aminoacid sequence of SEQ ID NO:2, and (b) binds to BMP2/4.
 13. Thepolypeptide of claim 12, wherein the polypeptide has at least 98%identity to the amino acid sequence of SEQ ID NO:2.
 14. The polypeptideof claim 12, wherein the polypeptide has at least 99% identity to theamino acid sequence of SEQ ID NO:2.
 15. A substantially purifiedpolypeptide comprising the amino acid sequence of SEQ ID NO:2 or afragment thereof, wherein the fragment is at least 40% of the length ofthe sequence shown as SEQ ID NO:2, and the fragment binds to BMP2/4. 16.A substantially purified polypeptide comprising the amino acid sequenceof SEQ ID NO:2 in which 5 or fewer amino acids are substituted, deleted,and/or inserted, and the polypeptide binds to BMP2/4.